Simultaneous analysis of Vitamins A and E in infant milk-based formulae by normal-phase high-performance liquid chromatography–diode array detection using a short narrow-bore column

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Abstract

A rapid, simple and reproducible normal-phase (NP) high-performance liquid chromatography (HPLC)–diode array detection (DAD) method for simultaneous qualitative and quantitative determination of Vitamin A (retinol acetate and retinol palmitate) and Vitamin E (α-tocopherol acetate, α-, γ- and δ-tocopherols) in milk-based infant formulae was developed and validated. The preparation sample was based on protein precipitation and vitamin extraction with ethanol, followed by re-extraction with hexane, while the chromatographic method was based on the use of a short narrow-bore column (50 mm × 2.1 mm; 3 μm particle size), which afforded less solvent consumption and higher mass sensitivity. The method showed acceptable values for precision, recovery and sensitivity, and proved very simple for routine analysis work.

Introduction

In general, Vitamin A refers to all-trans-retinol, which is the most active form of this vitamin, while Vitamin E is a collective term for tocopherols (α-, β-, γ- and δ-) and tocotrienols [1], [2]. Tocopherols and retinol are added to infant formulae (IF) both to improve the vitamin content and to prevent lipid oxidation during manufacture and storage, which helps to extend product shelf life. Moreover, IF also contain tocopherols originating from the oils used in their manufacture. The antioxidant activities of α-, β-, γ- and δ-tocopherols, those commonly found in vegetable oils, contribute more biologically active components to the diet than any other tocopherol isomer, although α-tocopherol is less stable in many products during manufacture and storage [3]. Therefore, fortification of IF with the more stable vitamin esters, such as α-tocopherol acetate, retinol acetate, or retinol palmitate is necessary [1], [2], [3], [4]. These molecules are more stable and less susceptible to oxidation. High-performance liquid chromatographic (HPLC) procedures have been of particular interest since initial reports of determination of Vitamin E in foods [4] have steadily risen. HPLC was first applied to the resolution of Vitamin E and other fat-soluble vitamins in 1971 by Schmit et al. [5]. Two reversed-phase packing materials, Permaphase ODS and Zipax HCP, were used to study the resolution of the fat-soluble vitamins, including α-tocopherol and α-tocopherol acetate. Permaphase ODS was a C18 column and Zipax HCP a hydrocarbon coating on Zipaz support. Van Niekerk [6] demonstrated the power of HPLC for vitamin analysis, and established important principles for the application of NP-HPLC to fat-soluble vitamins analysis: (1) as oils could be injected directly onto a silica column, no sample preparation other than dilution of the oil was required; (2) the positional isomers β- and γ-tocopherol could be resolved; (3) recovering added tocopherols to oils were high, approaching 100%. In addition, these procedures were “fast and easy”, with wide applicability for the routine assay of fat-soluble vitamins in foods [7], [8], [9], [10]. Detection of fat-soluble vitamins after HPLC resolution can be accomplished by UV (using diode array detection (DAD)), fluorescence (FLD), electrochemical (ED), or evaporative light scattering (ELSD) detection methods [11], [12], [13], [14], [15]. The most commonly used detector for Vitamins A and E analysis is FLD, which is considerably more sensitive and selective than UV, but less sensitive than ED.

NP-HPLC has been used to determine α-, β-, γ- and δ-tocopherols [16], [17], [18], [19] and retinol in IF [20], [21]. All of these procedures use FLD since FLD provides a sensitive and specific detection mode. For the simultaneous determination of Vitamins A and E, however, two different injections and mobile phases are necessary as FLD only works with one excitation wavelength (λex) and one emission wavelength (λem). For Vitamin E determination, this means that it is necessary to adjust the FLD configuration to λex = 285 nm, and λem = 310 nm; for Vitamin A determination, one must re-adjust the configuration to λex = 325 nm, and λem = 470 nm. On the other hand, DAD can work with multiple UV wavelengths, which are traduced with more versatility, but which are less sensitive for the detection of analytes compared with FLD.

The recent introduction of shorter narrow-bore columns (i.e. 50 mm × 2.1 mm; 3 μm particle size) in place of traditional columns (250 mm and/or 150 mm × 4.6 mm; particle size 5 μm) offers several advantages, including less solvent consumption and higher mass sensitivity [22]. An increase in mass sensitivity can permit the use of DAD to simultaneously analyze Vitamins A and E in IF in the same injection once these fat-soluble vitamins have been extracted.

Depending on the sample matrix, extraction of fat-soluble vitamins is usually performed by direct solvent extraction or saponification. Most oils that contain high levels of Vitamin E can be diluted with hexane or by mobile phase and directly injected into NP columns. This straightforward approach works well unless one component has low solubility in the mobile phase, or if other interfering compounds exist, such as sometimes occurs in IF. In this case, more extensive cleanup procedures must be employed. Preparation of the fat-soluble vitamin fraction for injection into the column from most food matrices requires either saponification of the sample matrix or a concentrated lipid fraction or extraction of total lipids from the sample, which can then be directly injected into a normal-phase column. Saponification converts the α-tocopherol acetate to α-tocopherol, and cannot be differentiated from the naturally occurring α-tocopherol [23]. In addition, saponification involves additional steps that are traduced when employed in increased-time analyses. Although there exist simplified methods involving saponification, it is reported that non-saponification of the total lipid dilution must be regarded as preferable to the saponification procedure as it offers higher accuracy [24]. Besides the elimination of the saponification step, this method permits the quantification of the added ester forms, as well as the natural Vitamins A and E homologues. The use of NP-HPLC makes fat removal unnecessary in IF, since it is known that the use of normal-phase silica allows the direct injection of oil; up to 2 mg per injection without influencing resolution, detection, or column life [16]. Finally, the stability of these fat-soluble vitamins is better both in the lipid matrix and with the ester forms of Vitamins A and E [21], [23], [24]. The solvent extraction method for fat-soluble vitamins used by Thompson and Hatina [16] has remained a standard for Vitamin E analysts for many years, although the extraction process is tedious. Moreover, the large solvent volumes required, and its attendant labor-intensiveness, renders this method less attractive. Solvents commonly used for fat-soluble vitamins extraction include chloroform–methanol (2:1), as employed in the Folch extraction method, acetone, diethyl ether, hexane, hexane–ethyl acetate, etc. [25].

The aim of this work was to develop and to validate an NP-HPLC–DAD method using a short narrow-bore column for simultaneously determining in a single injection α-tocopherol, α-tocopherol acetate, γ-tocopherol, δ-tocopherol, as well as retinol palmitate and retinol acetate, via a short, easy and straightforward direct-extraction method in IF.

Section snippets

Reagents and standards

The chemicals used for sample preparations were of analytical reagent grade. Hexane and ethyl acetate, both of HPLC-grade, were obtained from SDS (Peypin, France), absolute ethanol from Panreac (Barcelona, Spain), Standard of α-tocopherol acetate was obtained from Fluka (Buchs, Switzerland), and standards of α-, γ-, and δ-tocopherols and all-trans-retinol palmitate, and retinol acetate, were purchased from Sigma (St. Louis, MO, USA).

Instruments

We used a Hewlett-Packard liquid chromatographic system

Optimization of chromatographic conditions for Vitamins A and E separation

We first tested the mobile phases of 2 and 4% isopropanol in hexane using a pool of standards (retinol acetate, retinol palmitate, α-, γ-, δ-tocopherols and α-tocopherol acetate). Since complete separation of the peaks was not possible, we lowered the quantity of isopropanol, yielding 1.0 and 0.5%, but without obtaining any satisfactory results. Tan and Brzuskiewicz [27] optimized solvents systems for amino-, and silica-normal-phase columns using mobile phases consisting of 99% hexane and 1% of

Acknowledgements

The authors thank Laboratorios Ordesa S.L. (Sant Boi de Llobregat, Barcelona, Spain) for providing the samples. Special thanks go to CONACYT (Mexico) for supporting J.L.C.-S.

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